Interactions at the ligand binding domain (LBD) dimer interface of ionotropic glutamate receptors (iGluRs) play a pivotal role in receptor activation and desensitization. The current model of iGluR function proposes that the activation reaction starts with docking the agonist at a fixed LBD upper lobe (D1), followed by upward movement of the lower lobe (D2) towards D1 to lock the agonist within the LBD. A dimer interface between D1 lobes in adjacent subunits was identified in 3-D structures and is believed to help immobilize D1 and promote channel opening. In contrast, rupture of this interface, while agonist is bound, is thought to lead to an inactive, desensitized receptor conformation. While this proposed model for activation and desensitization describes macroscopic responses to a first approximation they have yet to be tested at the microscopic level. I propose to take advantage of the more advanced understanding of N-methyl-D-aspartate receptor (NMDAR) gating reaction to investigate the role in channel activation of residues at the dimer interface by recording both single- channel and whole-cell macroscopic currents from NMDARs with alterations within the LBD heterodimer interface. In addition to interactions common to all iGluRs, NMDARs also contain unique non-covalent interactions and an additional site II interface. Thus these studies will investigate mechanisms that are common to all iGluR as well as those that tell NMDARs apart. Contacts at the dimer interface will be modified by disulfide bond cross-linking and hydrophobic interactions, to immobilize the two D1-D1 lobes and presumably produce non-desensitizing receptors. Conversely, when contacts will be abolished by alanine substitution, the interface will be weakened and will produce receptors with faster and deeper desensitization. I also propose to examine key LBD heterodimer interface hotspots present in NMDARs but not in other iGluRs including site II contacts and D1-D2 interactions. The experiments outlined in this proposal will allow unprecedented insight into how the strength of intersubunit interface at the level of LBD contributes to NMDAR activation, deactivation and/or desensitization. They will also identify commonalities, as well as essential distinctions, between NMDAR and non- NMDAR activation mechanisms. The approach proposed is uniquely suited to dissect contributions to distinct gating transitions and to bridge the current gap in understanding between rearrangements at specific locations within iGluR structures and the overall functional result, usually evaluated with macroscopic current recordings. At present, the mechanistic approach proposed here is only possible for NMDARs, due to still inadequate understanding of non-NMDA iGluR microscopic kinetics. Results from these studies will illuminate mechanisms that are specific to NMDARs but also provide useful valuable insights into mechanisms that are common to all iGluRs. PUBLIC HEALTH RELEVANCE: NMDARs play a vital role in numerous physiological processes including neuronal development, synaptic strength and communication as well as neurological pathologies including excitotoxic cell death associated with stroke, schizophrenia, and neurodegeneration such as in Alzheimer's and Huntington's disease. The results of this proposal will help advance our understanding of excitatory transmission in the brain and of the ways in which this can be corrected to alleviate the burden of neurological diseases, addiction and mental illness.